RESUMO
Research into Schwann cell (SC)-related diseases has been hampered by the difficulty of obtaining human-derived SCs, which have limited proliferative capacity. This has resulted in a delay in progress in drug discovery and cell therapy targeting SCs. To overcome these limitations, we developed a robust method for inducing the differentiation of human induced pluripotent stem cells (hiPSCs) into SCs. We established hiPSC lines and successfully generated high-purity Schwann cell precursors (SCPs) from size-controlled hiPSC aggregates by precisely timed treatment with our proprietary enzyme solution. Such SCPs were successfully expanded and further differentiated into myelin basic protein (MBP) expressing SC populations when treated with an appropriate medium containing dibutyryl-cAMP (db-cAMP). These differentiated cells secreted factors that induced neurite outgrowth in vitro. Our method allows for the efficient and stable production of SCPs and SCs from hiPSCs. This robust induction and maturation method has the potential to be a valuable tool in drug discovery and cell therapy targeting SC-related diseases.
RESUMO
Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mesoderma/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genéticaRESUMO
Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5eGFP/w and MLC2vmCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5eGFP/w and MLC2vmCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Miosinas Cardíacas/metabolismo , Rastreamento de Células/métodos , Ventrículos do Coração/citologia , Proteína Homeobox Nkx-2.5/metabolismo , Miócitos Cardíacos/citologia , Cadeias Leves de Miosina/metabolismo , Células-Tronco Pluripotentes/citologia , Miosinas Cardíacas/genética , Diferenciação Celular/fisiologia , Separação Celular/métodos , Células Cultivadas , Técnicas de Introdução de Genes , Genes Reporter/genética , Ventrículos do Coração/metabolismo , Proteína Homeobox Nkx-2.5/genética , Humanos , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/genética , Células-Tronco Pluripotentes/metabolismo , Engenharia Tecidual/métodosRESUMO
VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.
Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Inativação Gênica , Genes de Partícula A Intracisternal/genética , Elementos Nucleotídeos Longos e Dispersos/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas , Metilação de DNA , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Espermatogênese/fisiologia , Testículo/metabolismoRESUMO
A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probeâ 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs. Among the KP-1-drug conjugates we synthesized, we found an exceptionally selective, chemically tractable molecule that induced the death of hiPSCs. Mechanistic analysis suggested that the high selectivity originates from the synergistic combination of transporter-mediated efflux and the cytotoxicity mode of action. The present study offers a chemical and mechanistic rationale for designing selective, safe, and simple reagents for the preparation of non-tumorigenic clinical samples.
Assuntos
Antineoplásicos/química , Separação Celular/métodos , Corantes Fluorescentes/química , Células-Tronco Pluripotentes Induzidas/citologia , Rodaminas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Corantes Fluorescentes/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Rodaminas/farmacologiaRESUMO
Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-ß 42 (Aß42)/Aß40 and Aß43/Aß40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Presenilina-1/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Embrionárias Humanas/patologia , Humanos , Mutação , Presenilina-1/genética , Regulação para CimaRESUMO
Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However, the current conventional method leads to poor recovery of hPSCs after thawing. Here, we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing, we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival, whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover, the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs.
Assuntos
Criopreservação/métodos , Laminina/fisiologia , Células-Tronco Pluripotentes/citologia , Quinases Associadas a rho/antagonistas & inibidores , Adesão Celular , Sobrevivência Celular , Crioprotetores , Meios de Cultura , Congelamento , Humanos , CariótipoRESUMO
One of the current obstacles to stem cell therapy is the tumorigenic potential of residual undifferentiated stem cells. The present study reports rediscovery of a synthetic derivative of okadaic acid, a marine polyether toxin, as a reagent that selectively induces the death of human pluripotent stem cells. Cell-based screening of 333 cytotoxic compounds identified methyl 27-deoxy-27-oxookadaate (molecule 1) as a substrate of two ATP-binding cassette (ABC) transporters, ABCB1 (MDR1) and ABCG2 (BCRP), whose expression is repressed in human embryonic stem cells and induced pluripotent stem cells. The results demonstrate that selective elimination of human pluripotent stem cells can be achieved by designing cytotoxic small molecules with appropriate ABC-transporter selectivity.
Assuntos
Produtos Biológicos/farmacologia , Ácido Okadáico/análogos & derivados , Ácido Okadáico/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Rodaminas/química , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Corantes Fluorescentes , Humanos , Neurônios/efeitos dos fármacosRESUMO
In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.
Assuntos
Cromatina/metabolismo , Ribonucleoproteínas/metabolismo , Espermatogênese , Animais , Cromossomos Artificiais Bacterianos , Masculino , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologiaRESUMO
AIMS: Development of a human cell-derived reentrant arrhythmia model is needed for studying the mechanisms of disease and accurate drug response. METHODS AND RESULTS: We differentiated human pluripotent stem cells (hPSCs) into cardiomyocytes, and then re-plated them into cell sheets that proved capable of forming electrically coupled assemblies. We monitored the function of these re-plated sheets optically with the Ca(2+) sensitive dye Fluo-4, and found that they generated characteristic waves of activity whose velocity and patterns of propagation depended upon the concentration of sodium channel blockers; lidocaine and tetrodotoxin, and also the time after re-plating, as well as the applied stimulation frequency. Importantly, reentrant spiral-wave propagation could be generated in these sheets by applying high-frequency stimulation, particularly when cell-density in the sheets was relatively low. This was because cardiac troponin T-positive cells were more non-homogeneously distributed at low cell densities. Especially in such sheets, we could terminate spiral waves by administering the anti-arrhythmic drugs; nifekalant, E-4031, sotalol, and quinidine. We also found that in these sheets, nifekalant showed a clear dose-dependent increase in the size of the unexcitable 'cores' of these induced spiral waves, an important parallel with the treatment for ventricular tachycardia in the clinical situation, which was not shown properly in cardiac-cell sheets derived from dissociated rodent hearts. CONCLUSIONS: We have succeeded in creating from hPSCs a valuable type of cardiomyocyte sheet that is capable of generating reentrant arrhythmias, and thus is demonstrably useful for screening and testing all sorts of drugs with anti-arrhythmic potential.
Assuntos
Arritmias Cardíacas/patologia , Modelos Cardiovasculares , Miócitos Cardíacos/patologia , Células-Tronco Pluripotentes/patologia , Engenharia Tecidual/métodos , Antiarrítmicos/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Desmossomos/ultraestrutura , Estimulação Elétrica , Humanos , Potenciais da Membrana/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Sarcômeros/ultraestrutura , Bloqueadores dos Canais de Sódio/farmacologia , Imagens com Corantes Sensíveis à Voltagem/métodosRESUMO
Epigenetic regulation is essential in determining cellular phenotypes during differentiation. Although tissue-specific DNA methylation has been studied, the significance of methylation variance for tissue phenotypes remains unresolved, especially for CpG-poor promoters. Here, we comprehensively studied methylation levels of 27 578 CpG sites among 21 human normal tissues from 12 anatomically different regions using an epigenotyping beadarray system. Remarkable changes in tissue-specific DNA methylation were observed within CpG-poor promoters but not CpG-rich promoters. Of note, tissue-specific hypomethylation is accompanied by an increase in gene expression, which gives rise to specialized cellular functions. The hypomethylated regions were significantly enriched with recognition motifs for transcription factors that regulate cell-type-specific differentiation. To investigate the dynamics of hypomethylation events, we analyzed methylation levels of the entire APOA1 gene locus during in vitro differentiation of embryonic stem cells toward the hepatic lineage. A decrease in methylation was observed after day 13, coinciding with alpha-fetoprotein detection, in the vicinity of its transcription start sites (TSSs), and extends up to â¼200 bp region encompassing the TSS at day 21, equivalent to the hepatoblastic stage. This decrease is even more pronounced in the adult liver, where the entire APOA1 gene locus is hypomethylated. Furthermore, when we compared the methylation status of induced pluripotent stem (iPS) cells with their parental cell, IMR-90, we found that fibroblast-specific hypomethylation is restored to a fully methylated state in iPS cells after reprogramming. These results illuminate tissue-specific methylation dynamics in CpG-poor promoters and provide more comprehensive views on spatiotemporal gene regulation in terminal differentiation.
Assuntos
Diferenciação Celular/fisiologia , Ilhas de CpG/fisiologia , Metilação de DNA/fisiologia , Regiões Promotoras Genéticas/fisiologia , Adulto , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Especificidade de Órgãos/fisiologiaRESUMO
Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose, development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF). Therefore, bFGF is thought to have an almost essential role for the expansion of hESCs in an undifferentiated state. Here, we report identification of small molecules, some of which were neurotransmitter antagonists (trimipramine and ethopropazine), which promote long-term hESC self-renewal without bFGF in the medium. The hESCs maintained high expression levels of pluripotency markers, had a normal karyotype after 20 passages, and could differentiate into all three germ layers.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Compostos Orgânicos/farmacologia , Bibliotecas de Moléculas Pequenas , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Metotrimeprazina/química , Metotrimeprazina/farmacologia , Camundongos , Camundongos SCID , Estrutura Molecular , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Compostos Orgânicos/química , Fenotiazinas/química , Fenotiazinas/farmacologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Prometazina/química , Prometazina/farmacologia , Trimeprazina/química , Trimeprazina/farmacologia , Trimipramina/química , Trimipramina/farmacologiaRESUMO
Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.
Assuntos
Adenoviridae/genética , Células-Tronco Embrionárias/metabolismo , Vetores Genéticos/genética , Recombinação Homóloga , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos Nucleares/genética , Linhagem Celular , DNA Ligase Dependente de ATP , DNA Ligases/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Ordem dos Genes , Marcação de Genes , Heterozigoto , Humanos , Hipoxantina Fosforribosiltransferase/genética , Células-Tronco Pluripotentes Induzidas/citologia , Autoantígeno Ku , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas de XenopusRESUMO
Chromeceptin is a synthetic small molecule that inhibits insulin-induced adipogenesis of 3T3-L1 cells and impairs the function of IGF2 (insulin-like growth factor 2). The molecular target of this benzochromene derivative is MFP-2 (multifunctional protein 2). The interaction between chromeceptin and MFP-2 activates STAT6 (signal transducer and activator of transcription 6), which subsequently induces IGF inhibitory genes. It was not previously known how the binding of chromeceptin with MFP-2 blocks adipogenesis and activates STAT6. The results of the present study show that the chromeceptin-MFP-2 complex binds to and inhibits ACC1 (acetyl-CoA carboxylase 1), an enzyme important for the de novo synthesis of malonyl-CoA and fatty acids. The formation of this ternary complex removes ACC1 from the cytosol and sequesters it in peroxisomes under the guidance of Pex5p (peroxisomal-targeting signal type 1 receptor). As a result, chromeceptin impairs fatty acid synthesis from acetate where ACC1 is a rate-limiting enzyme. Overexpression of malonyl-CoA decarboxylase or siRNA (small interfering RNA) knockdown of ACC1 results in STAT6 activation, suggesting a role for malonyl-CoA in STAT6 signalling. The molecular mechanism of chromeceptin may provide a new pharmacological approach to selective inhibition of ACC1 for biological studies and pharmaceutical development.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/metabolismo , Benzopiranos/química , Benzopiranos/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células Hep G2 , HumanosRESUMO
Although human pluripotent stem cell (hPSC) lines were initially established in culture using feeder cells, the development of culture media and substrates is essential for safe, stable, high-quality, and efficient production of large numbers of cells. Many researchers are now culturing hPSCs in chemically defined media and on culture substrates without feeder cells. In this review, we first discuss the problems with Matrigel, which has long been used as a culture substrate. Then, we summarize the development of extracellular matrix proteins for hPSCs, which are now the mainstream alternative, and synthetic substrates that are expected to be the future mainstream alternative. We also highlight three-dimensional culture for suitable mass production of hPSCs.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Pluripotentes , Humanos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Células Alimentadoras , Meios de Cultura/metabolismo , Diferenciação CelularRESUMO
The pluripotency factor Nanog is expressed in peri-implantation embryos and primordial germ cells (PGCs). Nanog-deficient mouse embryos die soon after implantation. To explore the function of Nanog in germ cells, Nanog RNA was conditionally knocked down in vivo by shRNA. Nanog shRNA transgenic (NRi-Tg) mice were generated through the formation of germline chimeras with NRi-Tg embryonic stem cells. In E12.5 Cre-induced ER-Cre/NRi-Tg and TNAP-Cre/NRi-Tg double-transgenic embryos, the number of alkaline phosphatase-positive and SSEA1-positive PGCs decreased significantly. In the E9.5 and E10.5 migrating Nanog-knockdown PGCs, TUNEL-positive apoptotic cell death became prominent in vivo and in vitro, despite Oct4 expression. Single-cell microarray analysis of E10.5 Nanog-knockdown PGCs revealed significant up- and downregulation of a substantial number of genes, including Tial1, Id1 and Suz12. These data suggest that Nanog plays a key role in the proliferation and survival of migrating PGCs as a safeguard of the PGC-specific molecular network.
Assuntos
Apoptose/genética , Células-Tronco Embrionárias/metabolismo , Células Germinativas/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Animais , Morte Celular/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Vetores Genéticos , Células Germinativas/citologia , Células Germinativas/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Integrases/genética , Integrases/metabolismo , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Homeobox NanogRESUMO
BACKGROUND: Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata. RESULTS: Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI) nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells. CONCLUSIONS: PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations.
Assuntos
Diferenciação Celular , Ouro/química , Mamíferos/metabolismo , Nanopartículas Metálicas/química , Sequência de Aminoácidos , Animais , Adesão Celular , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Imidas/química , Nanopartículas Metálicas/ultraestrutura , Camundongos , Dados de Sequência Molecular , Miócitos Cardíacos/citologia , Nanofibras/ultraestrutura , Tamanho da Partícula , Peptídeos/química , Poliestirenos/químicaRESUMO
Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency.
Assuntos
Células-Tronco Embrionárias/metabolismo , Marcação de Genes/métodos , Loci Gênicos , Transgenes , Linhagem Celular , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica , Humanos , Hipoxantina Fosforribosiltransferase/genética , Recombinação GenéticaRESUMO
The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Algoritmos , Animais , Biologia Computacional/tendências , Bases de Dados de Proteínas , Perfilação da Expressão Gênica/métodos , Genoma de Planta , Genoma Viral , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Japão , SoftwareRESUMO
Embryonic stem cells (ESCs) are useful resources for drug discovery, developmental biology and disease studies. Cellular microenvironmental cues play critical roles in regulating ESC functions, but it is challenging to control them with synthetic components. Nanofibers hold a potential to create artificial cellular cues for controlling cell adhesion and cell-cell interactions. Mouse ESC (mESC) were cultured on electrospun nanofibers made from polymethylglutarimide (PMGI), which is a synthetic thermoplastic polymer stable under culture conditions. Both topology and the density of PMGI nanofibers were key factors. mESCs on nanofibers had a growth rate comparable to those cultured conventionally and retained their pluripotency. Furthermore, self-renewed ESCs differentiated into all three germ layers thereby providing a reliable way to expand mESCs without feeder cells.